Insulin facilitates glucose uptake into adipocytes and muscle cells by relocating glucose transporter 4 {GLUT4) from intracellular reservoirs to the plasma membrane. The translocation involves a vesicle fusion step that is mediated by three SNAREs - syntaxin 4, SNAP-23 and VAMP2 - and a number of regulatory proteins including MunciSc, synip and tomosyn. While the physiological importance of the SNARE regulatory proteins is clear, their molecular mechanisms of action and functional interactions among themselves are not known due to the complexity of the cellular environment. Recently, we reconstituted the SNARE-dependent GLUT4 vesicle fusion in both liposome (synthetic bilayers) and "flipped" SNARE cell-cell (native membranes) fusion systems. Here we propose to capitalize on these unique developments to ask key mechanistic questions about GLUT4 vesicle fusion, especially questions concerning how regulatory proteins act, alone or in concert, to control exocytosis at the molecular level. The specific hypothesis behind this proposed research is that regulatory proteins control different stages of the SNARE assembly cycle and contribute to the temporal and spatial Vegulation of GLUT4 exocytosis. Regulatory proteins will be added either as pure recombinant proteins or expressed as flipped proteins on th& cell surface. Kinetic effects of each regulator can be assessed when the regulator is added (alone or in combination) to the core fusion machinery of SNAREs. Two specific aims are proposed: 1) Define how the concerted action of SNAREs and regulatory factors controls GLUT4 vesicle fusion; 2) Characterize the fusion pore dynamics and transition state of GLUT4 exocytosis in the "flipped" SNARE fusion system. Our long-temn goal is to work our way up, protein by protein, until we can reconstitute the basic properties and fine-tuning of GLUT4 exocytosis. Insulin-regulated GLUT4 transport is crucial for glucose homeostasis, and imbalances in this process may lead to type 2 diabetes. Knowledge of how SNARE regulators work may Identify novel targets for therapeutic intervention. Since many components of GLUT4 transport are conserved, our work can also dhed light upon other exocytic pathways such as platelet and lung epithelial secretion.